1. Field of the Invention:
The present invention relates to a process for rapidly estimating the amount or presence of stable-type glycated hemoglobin in a sample, and to an apparatus for use in clinical tests to run said process.
2. Discussion of the Background:
Glycated hemoglobin is produced when a saccharide in blood, mostly glucose, enters an erythrocyte and combines with hemoglobin. These saccharides enter erythrocytes in an amount proportional to their concentration in blood.
The concentration of glycated hemoglobin in a patient reflects the mean concentration of glucose in the patient's blood through a few past months. Since the amount of glycated hemoglobin is less susceptible to physiological factors than is glucose in blood and urine, it is a more suitable index for assays used in the diagnosis of diabetes and the follow-up observation of diabetic patients.
The main component of glycated hemoglobin is a hemoglobin having attached thereto glucose at the N-terminal of the hemoglobin .beta. chain. This combination is performed in two steps involving a non-enzymatic reaction.
In the first step of the reaction the glycated hemoglobin is formed reversibly and partly decomposes to hemoglobin and saccharide. This forms so-called unstable-type glycated hemoglobin.
The second reaction is an irreversible reaction in which stable-type glycated hemoglobin is formed. A more reliable index of diabetes is obtained of course by measuring the amount of the stable-type glycated hemoglobin.
Glycated hemoglobin can be separated from hemoglobin by utilizing the differences in the electrical properties of these two materials. Methods useful for this separation include electrophoresis and ion exchange chromatography. An apparatus of high performance liquid chromatography for this particular purpose is also commercially available.
Glycated hemoglobin can be separated from ordinary hemoglobin using these methods alone, but glycated hemoglobins in their stable and unstable-types cannot be distinguished from each other. Therefore, to obtain an estimation of the stable-type glycated hemoglobin a prior treatment for the removal of the unstable-type glycated hemoglobin is required.
Prior available methods include a process in which erythrocytes are incubated in a physiological saline solution or in a buffer solution containing semicarbazide and aniline. Another process is available in which whole blood is hemolyzed in a hemolysis solution containing boric acid before incubation.
With the method which uses a physiological saline solution treatment, the erythrocytes need to be washed several times with the physiological saline solution prior to incubation, and subsequent incubation at 37.degree. C. for more than 4 hours is required (P. A. Svensen et al., Diabetologia, 19, 130 (1980)). On the other hand, the semicarbazide--aniline method involves unstable reagent solutions which need to be freshly prepared immediately before use, and subsequent incubation for 30 min to an hour at 37.degree. C. is also required (D. M. Nathan et al., Clin. Chem., 28, 512 (1982)).
On the other hand, commercial reagents are available with which the unstable-type glycated hemoglobin can be removed. And though no problem is involved with respect to their stability, processes using these reagents require an incubation time of about 30 min at 37.degree. C. as in the preceeding example.
All available methods suffer the inconvenience of requiring troublesome pre-treatment operations or problems preventing the rapid treatment of samples. There is therefore a strongly felt need for a new process for rapidly assaying stable-glycated hemoglobin in a patient and an apparatus for performing such a clinical test.